The efforts of the Molecular Mechanisms of Tumor Promotion Section are directed at understanding the early events in the interaction of phorbol ester tumor promoters with cells and tissues. Particular attention is being devoted to the analysis of the major phorbol ester receptor, protein kinase C. Novel protocols have been developed for its purification that are more efficient and afford higher yields than is possible with current methods. Preparation of monoclonal and polyclonal antibodies to the receptor is in progress. The role of lipids in reconstitution of the receptor has been characterized in detail. Phospholipids differ in whether or not they can reconstitute in the amounts required for reconstitution, and in the phorbol ester structure-activity relations of the resultant complex. Diacylglycerols competitively inhibit phorbol ester binding in vitro, consistent with their being the postulated endogenous phorbol ester analogs. Comparison with the homologous phorbol esters yields differences in affinities of 20- to 30,000-fold, depending on the derivative, implying distinct side chain requirements for phorbol esters and diglycerides. As expected from the in vitro assays, treatment of intact cells with phospholipase C to generate diacylglycerol endogenously or the exogenous addition of appropriate diacylglycerols likewise inhibits phorbol ester binding competitively in vivo and induces phorbol ester-like responses. Identification of the enzymatic activity associated with the phorbol ester receptor has made it possible to analyze the coupling between binding and subsequent response. Most mouse skin tumor promoters, structurally unrelated to the phorbol esters, did not activate protein kinase C in vitro. Unsaturated fatty acids at high concentrations did activate, however. Multiple phorbol ester receptors have been implicated in the heterogeneity of phorbol ester responses. The binding characteristics of intact, cultlured keratinocytes change from homogeneous to heterogeneous as differentiation proceeds.